Human cystolic phospholipase A.sub.2 (cPLA) is an arachidonic acid specific enzyme which is of considerable interest due to its proposed roles in arachidonic acid release, eicosanoid production, and signal transduction.
This enzyme bears little resemblance to the more well-studied extracellular or secretory phospholipase A.sub.2 enzymes (sPLA) obtained from snake or bee venom, mammalian pancreas, or human synovial fluid. The sPLA's are small, 14 kDa proteins which are dependent on Ca.sup.2+ for catalytic activity.
The cPLA enzyme is an 85 kDa protein which is also activated by Ca.sup.2+ (Clark et al. Cell 65:1043-1051 (1991) and Sharp et al., J. Biol. Chem. 266:14850-14853 (1991)). However, rather than playing a catalytic role, the Ca.sup.2+ appears to be required for binding of the enzyme to the membrane substrate (Clark et al. supra; Wijkander et al., Biochem. Biophys. Res. Comm. 184:118-124 (1992); Ghomashchi et al., Biochemistry 31:3814-3824 (1992); and Reynolds, et al., Biochim. Biophys. Acta 1167:272-280 (1993)). Another difference between the enzymes is that the sPLA's catalyze only the phospholipase A.sub.2 reaction while cPLA is non-specific and displays lysophospholipase and transacylase activities as well as phospholipase A.sub.2 activity (Reynolds, et al., supra).
Because of the potential role of this enzyme in eicosanoid production, there is considerable interest in developing pharmacologically effective inhibitors of cPLA as anti-inflammatory agents. Accordingly, assays that are capable of detecting cPLA are necessary for designing inhibitors of the enzyme.
The assays currently utilized for the measurement of cPLA enzyme are radioactive assays which involve organic extraction of lipids, usually by either the methods of Dole, (Ibrahim, S. S. Biochim. Biophys. Acta 137:413-419 (1967)); or Bligh and Dyer, (Can. J. Biochem. Biophysiol. 37:911-917 (1959)); followed by either a silica column or TLC separation.
These radioactive assays utilize a variety of substrate forms including pure sonicated phospholipid vesicles, (Ghomashchi et al., supra; and Leslie, C. C. J. Biol. Chem. 266:11366-11371 (1991)); sonicated phospholipid/dioleoylglycerol (DAG) mixtures, (Kramer, R. M., et al. J. Biol. Chem. 266:5268-5272 (1991)); and mixed micelles of phospholipid/Triton X-100 with glycerol, (Clark, J. D., et al. Proc. Natl. Acad. Sci. 87:7708-7712 (1990), and Ulevitch, R. J. et al. J. Biol. Chem. 263:3079-3085 (1988)). These radioactive assays are generally tedious and difficult to use in routine screening programs. They also each have particular drawbacks. Most notable is a non-linear time course observed in vesicle assays, where the activity plateaus and stops after only a few minutes. The DAG assay, while not linear, has a somewhat better time course, but is typically run at a very low substrate concentrations where the interpretation of inhibition data can be difficult. The Triton/glycerol assays have the best time course, but require a considerable amount of enzyme.
Accordingly, it is an object of this invention to provide a novel substrate for cPLA that can be used in a spectrophotometric assay. It is a further object of this invention to provide a rapid, convenient, and reliable assay for cPLA that exhibits a linear time course. Additional objects and features of the invention will be apparent to those skilled in the art from the following detailed description and appended claims when taken in conjunction with the figures.